buffer and molarity.. choice? - pH 7.0 (Dec/27/2006 )
heyy!
i'm to use a buffer around pH 7.0. i have a choice of tris and phosphate.. which one shud i choose? what molarity wud be enough.. molarity increases.. buffering capacity increases, right?
my purpose is to use the buffer for cation exchange chrom for a protein.
basically, for any experiment.. how will u choose the buffer and the molarity of it to be used?? plsss lemme know!
thankuuuuuuuu!
Well, this is usually determined by the properties of the buffer other than its buffering ability. For example, if you are working with a calcium containing solution, phosphate would be a poor buffer choice because of the precipitation of calcium phosphate. If you are working with RNA, then DEPC treatment of Tris would not be possible, so Tris might be a bad choice. Similarly, if temperature stability of the pH was a goal, the poor temperature coefficient of Tris would be a problem. Given a choice, I would think that in general HEPES or MOPS would be very good choices for pH 7.0 buffers, but it really is application dependent. Buffer capacity does increase with molarity, but molarity can have other effects, such as direct effect on the osmolarity of the solution. You'd like your buffer concentrations to be higher than the concentrations of the compounds you are trying to buffer against.
As far as choice of sodium or potassium phosphates, the answer again is that it is application dependent. Since in most cases these ions will be completely dissociated, you are really asking what other ion can I put in solution which will not mess up my experiment. Often this is tradition, or affected by solubility. For example, mixing potassium ions into a solution containing SDS will precipitate potassium dodecyl sulfate, whereas SDS is quite soluble. Tradition usually suggests sodium as a counter ion unless there is reason for another choice.
i'm to use a buffer around pH 7.0. i have a choice of tris and phosphate.. which one shud i choose? what molarity wud be enough.. molarity increases.. buffering capacity increases, right?
my purpose is to use the buffer for cation exchange chrom for a protein.
basically, for any experiment.. how will u choose the buffer and the molarity of it to be used?? plsss lemme know!
thankuuuuuuuu!
in principle you can use each for cationic exchanger chromatography; if you cool your chromatography system you may get difficulties with phosphate precipitations if you use a Pi-buffer (f.i. if your extract contains considerable amounts of Calcium); so, in this case I would prefer Tris
although buffer capacity rise with molarity of buffering component, you should not choose a too high conc because of ionic strength effects; good ranges are between 20 and 50 mM for chromatography
on the other hand, pH 7.0 is out of the effective buffering range of tris but in the range of phosphate. so, given the choices you have, i would select the phosphate buffer.